中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (46): 8703-8708.doi: 10.3969/j.issn.2095-4344.2012.46.031

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

抗极低密度脂蛋白受体单克隆抗体的大量制备及纯化

杨 璞1, 2，李 琦2, 3，刘志国2, 3，田 俊2，宗义强2，屈 伸2   

  1. 1华南理工大学生物科学与工程学院生物医学工程系，广东省广州市 510006
    2华中科技大学同济医学院基础医学院生物化学与分子生物学系，湖北省武汉市 430030
    3武汉工业学院生物与制药工程学院，湖北省武汉市430023
  • 收稿日期:2012-04-05 修回日期:2012-04-24 出版日期:2012-11-11 发布日期:2012-11-11
  • 通讯作者: 屈伸，教授，华中科技大学同济医学院基础医学院生物化学与分子生物学系，湖北省武汉市 430030 qushen@mails.tjmu.edu.cn
  • 作者简介:杨璞☆，女，1980年生，湖北省老河口市人，汉族，2008年华中科技大学同济医学院毕业，博士，主要从事动脉粥样硬化的分子机制研究。 yangpu254@yahoo.com.cn

Preparation and purification of a great amount of monoclonal antibodies against the c-terminal of very low density lipoprotein receptor

Yang Pu1, 2, Li Qi2, 3, Liu Zhi-guo2, 3, Tian Jun2, Zong Yi-qiang2, Qu Shen2   

  1. 1Department of Biomedical Engineering, School of Biological Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong Province, China
    2Department of Biochemistry and Molecular Biology, Basic Medical School, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430030, Hubei Province, China
    3School of Biotechnology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, Hubei Province, China
  • Received:2012-04-05 Revised:2012-04-24 Online:2012-11-11 Published:2012-11-11
  • Contact: Qu Shen, Professor, Department of Biochemistry and Molecular Biology, Basic Medical School, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430030, Hubei Province, China qushen@mails.tjmu.edu.cn
  • About author:Yang Pu☆, Ph.D., Department of Biomedical Engineering, School of Biological Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong Province, China; Department of Biochemistry and Molecular Biology, Basic Medical School, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430030, Hubei Province, China yangpu254@yahoo.com.cn

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416

被引次数

8

摘要：

背景：极低密度脂蛋白受体被认为是一种“瑞士军刀”样多功能受体，对该受体的研究很广泛，但其商品化抗体种类较少，价格偏贵。
目的：获得研究可用的抗极低密度脂蛋白受体的单克隆抗体。
方法：为了获得抗极低密度脂蛋白受体单克隆抗体，选用可分泌针对极低密度脂蛋白受体胞内段的单克隆抗体的杂交瘤细胞IgG 6A6。通过将杂交瘤细胞IgG 6A6注入Balb/c小鼠腹腔内，2周后收集腹水，分别通过盐析法(正辛酸-饱和硫酸铵沉淀法)初步纯化和亲和层析进一步纯化目的蛋白，获得针对极低密度脂蛋白受体胞内段的单克隆抗体。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测其相对分子质量和纯度，酶联免疫吸附实验检测其活性。
结果与结论：未纯化腹水Western Blotting结果显示条带较多，背景不干净；而经正辛酸-饱和硫酸铵法初步纯化的腹水、经Protein A进一步纯化得到的抗体均可特异识别极低密度脂蛋白受体。提示经盐析法和Protein A Agarose纯化可获得较纯和有活性的单克隆抗

关键词: 极低密度脂蛋白受体, 单克隆抗体, 纯化, 盐析法, 亲和层析

Abstract:

BACKGROUND: Very low density lipoprotein receptor is regarded as a Swiss Army Knife receptor, and many researchers have studied this receptor. But there are only several kinds of commercial antibodies and the price is on the high side.
OBJECTIVE: To get the monoclonal antibody against the c-terminal of very low density lipoprotein receptor for the future study.
METHODS: In order to obtain the very low density lipoprotein receptor, hybridoma IgG 6A6 cells related with very low density lipoprotein receptor intracellular domain was injected into peritoneal of Balb/c mice. After 2 weeks, ascites was collected, and the target protein was preliminary purified by salting-out method (n-octanoic acid-saturated ammonium sulfate precipitation method) and further purified by affinity chromatography, and the monoclonal antibody related with very low density lipoprotein receptor intracellular domain was obtained. The relative molecular mass and purity were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the activity was detected by enzyme-linked immunosorbent assay.
RESULTS AND CONCLUSION: Western Blotting results showed that the antibody from the ascites directly can bind the very low density lipoprotein receptor protein, but the background was not clear. The purified antibody from the ascites through salting-out method (n-octanoic acid-saturated ammonium sulfate precipitation method) and then by protein A affinity chromatography can bind specifically with the very low density lipoprotein receptor. The pure and activity monoclonal antibodies can be obtained through purification by the salting-out method and Protein A Agarose

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